New recommendations for the standardization of research on endothelial precursor cells are expected to optimize future investigations of these cells in systemic sclerosis and to simplify the comparison of different studies, according to the authors.
Endothelial precursor cells (EPCs) play an important role in the homeostasis of the vascular network and are considered potential candidates for novel therapeutic approaches as well as possible biomarkers for vascular repair, new vessel formation, and cardiovascular prognosis. However, methodical and other inconsistencies across research studies complicate data interpretation, wrote Dr. Jörg H.W. Distler of the University of Elrangen-Nuremberg (Germany) and colleagues in the European League Against Rheumatism Scleroderma Trials and Research (EUSTAR) group. Specifically, different protocols for EPC isolation, enrichment, culture, and quantification, as well as insufficient data on potentially confounding risk factors, have led to conflicting results in previous studies (Ann. Rheum. Dis. 2009;68:163–8).
Among the holes in the current pool of research is the absence of studies demonstrating EPCs in vascular lesions of animal models of systemic sclerosis, the authors wrote, noting that, to date, studies have shown EPCs in vascular lesions of ischemia. Additionally, “the mechanisms by which EPCs have contributed to vascular repair and neovascularization have not fully been elucidated,” they wrote. “It remains to be determined whether EPCs mediate their effects in humans independently from mature endothelial cells or whether EPCs function more as bystanders of angiogenesis.”
Finally, the numbers of EPCs characteristically in the blood of patients with systemic sclerosis is in need of clarification, as the results of existing studies are contradictory. “The initial study suggested a profound decrease of circulating EPCs, whereas subsequent studies found increased numbers of EPCs in patients with systemic sclerosis”—differences that might be a function of different disease durations in the study patients or different cell enrichment techniques prior to fluorescence-activated cell sorting (FACS) analysis, the authors wrote.
The following recommendations should be incorporated in future studies, the authors advised:
▸ Studies should include a detailed description of methods and materials used.
▸ Cardiovascular risk factors and drugs should be described in detail. Statins, in particular, impact circulating EPCs.
▸ Studies with small numbers of patients should be avoided because they are of limited help in light of the heterogeneity of systemic sclerosis and the large number of potential confounding factors that influence the number of EPCs.
▸ Basic methodological guidelines for the isolation, culture, enrichment, and detection of EPCs should be followed and described in detail.
▸ For in vitro EPC culture, the contents of endothelium growth medium 2 (EGM-2) are best defined and as such should be the medium of choice for future experiments.
▸ Culture dishes should be coated with laminin and type IV collagen because of the close resemblance to the vascular basal membrane.
▸ For all in vitro cultures, the endothelial phenotype should be confirmed at the end of the culture period.
▸ For the quantification of EPCs in the blood, the expression of CD133, vascular endothelial growth factor type 2 receptor (VEGFR2), and CD34 together with a viability marker should be evaluated in a multiparameter flow cytometer.
▸ Standard operating procedures for FACS (see box) must be strictly followed.
The various authors reported receiving research grants and/or honoraria from several major pharmaceutical companies.
Flow Cytometry for EPC Detection
Strict adherence to the following standard operating procedures for fluorescence-activated cell sorting (FACS) analysis in endothelial cell precursor research is critical, according to the EUSTAR statement authors.
▸ Clean the flow cytometer rigorously to avoid sample contamination.
▸ Set and monitor the sensitivity of fluorescence detectors.
▸ Collect a minimum of 500,000 events to collect an adequate number of endothelial precursor cells.
▸ Use a real-time viability stain, such as 7AAD or propidium iodide, and identify and exclude dead cells to minimize nonspecific staining and improve assay resolution.
▸ Use a blocking serum to decrease nonspecific binding via Fc receptors.
▸ Establish a dump channel in order to exclude from analysis those cells that are not of interest.